The best ratio for any new antibody reagent must be determined by experimentation but 80-100µg of IgG antibody for every 100µg of GOx usually gives optimal results. The 100µg quantity of antibody corresponds to an Antibody:GOx molar ratio of 1:1 (Mw of GOx is 160kDa).

The volume in which the antibody is added ideally should be between 40 and 100µl.

Protocol

Before you add antibody to the GOx mix, add 10µl of Modifier reagent for each 100µl of antibody to be labeled. Mix gently.

Remove the screw cap from the vial of GOx mix and pipette the antibody sample (with added Modifier) directly onto the lyophilised material. Resuspend gently by withdrawing and re-dispensing the liquid once or twice using a pipette.

Place the cap back on the vial and leave the vial standing for 3 hours in the dark at room temperature (20-25°C). Alternatively, and often more conveniently, conjugations can be set up and left overnight, as the longer incubation time has no negative effect on the conjugate.

After incubating for 3 hours (or more), add 1µl of Quencher reagent for every 10µl of antibody used. The conjugate can be used after 30 minutes.

Storage of conjugates

For any new conjugate, initial storage at 4°C is recommended. A preservative may be desirable for long-term storage. Other storage conditions (e.g. frozen at -70°C or stored at -20°C with 50% glycerol) may also be satisfactory. The best conditions for any particular conjugate must be determined by experimentation.

Sample Buffer

Ideally, the antibody to be labeled should be in 10-50mM amine-free buffer pH range 6.5 to 8.5. However, many buffers outside these limits of concentration and pH can be accommodated. Modest concentrations of Tris buffer are also tolerated.

Amine-free buffers, including MES, MOPS, HEPES and phosphate are compatible if they are in the concentration range 10-50mM and have pH values in the range 6.5-8.5, as the addition of Modifier provides the conditions necessary for efficient conjugation. Common non-buffering salts (e.g. sodium chloride), chelating agents (e.g. EDTA), and sugars may be present, as they have no effect on conjugation efficiency. Azide (0.02-0.1%) has little or no effect. If the amine-free buffer is relatively concentrated and outside the pH range 6.6-8.5 you may need to add more Modifier for each 10µl of antibody. Excess Modifier is provided so that you can check the pH of the buffer after the addition of the modifier. Ideally the pH should be around 7.3-7.6, though efficient conjugation occurs anywhere between pH 6.8 and 7.8. Avoid buffer components that are nucleophilic, as these may react with the kit chemicals. If your buffer contains primary amines (e.g. amino acids, ethanolamine) and/or thiols (e.g. mercaptoethanol, DTT), you should consider using our Concentration and Purification Kits (ab102778 or ab102784). (Note: Unusually, for an amine, Tris has little effect on conjugation efficiency as long as the concentration is 20mM or less).

Disclaimer

Labeling of the antibody will not work if the conjugation blocks the active paratope. This situation is rare.

The antibody to be labelled should be purified, in an appropriate buffer for conjugation and at a suitable concentration, as described in the protocol booklet. If not, consider using our antibody purification and concentration kits.